Prevailing experimental evidence suggests that counter-regulatory crosstalk between TGFbeta/BMP/Smad4 and Wnt/beta-catenin signaling pathways is central to normal intestinal homeostasis and that loss of this regulation promotes carcinogenesis, however, the underlying mechanisms of this regulatory crosstalk are incompletely understood. The long-term goal of our laboratory is to delineate the mechanisms that drive colorectal carcinogenesis and tumor progression and to identify improved prognostic biomarkers and novel therapeutic targets in colon cancer. The overall objective of this proposal is to identify specific interactions between Smad4 and Wnt signaling pathways that maintain homeostasis and drive tumor cell behavior in the intestine. Based upon our own work and that from other laboratories, we propose to test the following central hypothesis: loss of Smad4 expression in colon epithelial cells and colon cancer cells amplifies &#946;-catenin expression and Wnt signaling, thereby promoting tumor progression. The rationale underlying the proposed research is based on our observations that Smad4 loss in colonic crypts leads to disruption of intestinal homeostasis and enhanced susceptibility to intestinal tumorigenesis and that silencing of Smad4 in cell lines results in increased beta-catenin expression and increased beta-catenin/TCF activated gene expression. The goal of this proposal is to determine the biological consequences of Smad4 loss in the intestine and to determine the mechanism by which Smad4 loss increases beta-catenin and amplifies Wnt signaling. Specific Aim 1: Determine the impact of intestinal epithelial cell loss of Smad4 signaling on Wnt signaling, homeostasis and barrier function in the mouse intestinal tract. Working hypothesis: Smad4 regulation of beta-catenin mRNA expression in the intestinal tract is required for homeostasis and barrier function in the intestinal tract. We will use innovative mouse models with inducible tissue specific depletion of Smad4 to test this working hypothesis. Specific Aim 2: Determine the mechanism of Smad4-mediated regulation of beta-catenin mRNA expression and beta-catenin activated gene expression. Working Hypothesis: The tumor suppressive activity of Smad4 occurs through repression of &#946;-catenin expression and inhibition of the beta-catenin dependent transcriptional program. In this aim, we will determine the mechanisms by which Smad4 mediated signaling impacts beta-catenin expression and Wnt/&#946;-catenin activated gene expression. This proposal has high impact because it fills significant gaps in understanding of how an important tumor suppressor and proto-oncogene interact to achieve homeostasis in the normal intestinal epithelium, and how disruption of this normal regulation leads to metastasis and mortality in cancer. Through these insights, we expect to identify novel targets for therapeutic intervention.